Summary The goal of this project is to investigate a special type of intercellular communication common in animal germline cells. From flies to humans, the development of eggs and sperm is carried out in groups of germline cells connected by intercellular bridges that allow the exchange of cytoplasm. Disruption of intercellular exchange causes infertility. These intercellular bridges, called ring canals, arise from mitotic cleavage furrows between cells that do not complete cytokinesis. Ring canals are also present in several somatic epithelial tissues in the fruit fly Drosophila, providing evidence that stable intercellular bridges are relevant to non- germline biology. The experiments are designed to elucidate the composition, regulation and function of ring canals using the powerful genetic and cell biological approaches in the Drosophila model system. The three integrated aims of this proposal will greatly enhance the understanding of ring canal formation and regulation, identify new ring canal components, and examine the function of ring canals in the male germline and somatic tissues. Research proposed in Aim 1 will investigate the regulation of the actin cytoskeleton of female germline ring canals, which is responsible for the expansion of ring canals that is needed to accommodate movement of maternal cytoplasm to the growing oocyte. Recent work in the lab uncovered an unusual regulatory mechanism for controlling the organization of filamentous actin that involves protein degradation via the ubiquitin proteasome system and the activity of an E3 ubiquitin ligase. Proteomic and biochemical approaches will be used to identify substrates of the ligase and characterize their role in F-actin assembly at the ring canal. Since the E3 ubiquitin ligase involved with organizing ring canal F-actin is highly conserved, these experiments will be of great interest to researchers studying regulated protein degradation. Experiments in Aim 2 will quantify the largely uncharacterized cytoplasmic exchange occurring in both germline and somatic ring canals in the male reproductive tract. To elucidate the functional importance of ring canals, novel strategies involving targeted destruction or occlusion of ring canals will be carried out. The results will reveal the importance of communication and molecular exchange through ring canals in these tissues. Newly optimized proteomic methods proposed in Aim 3 involve in vivo chemical labeling with a biotin tag and extraction of intact ring canals or ring canal components. The proteomes of germline and somatic ring canals in male and female reproductive tissues will be compared, allowing new insight into their composition and the conserved features of ring canals. The comparative proteomics approach will also reveal new ring canal constituents to target for genetic and functional analysis. Another approach will use the same tools to probe localized protein environments, rather than overall composition, and will yield unprecedented information on the relationships of components within the structure of the ring canal cytoskeletal network.